Beclin 2 functions in autophagy, degradation of G protein-coupled receptors, and metabolism.

The molecular mechanism of autophagy and its relationship to other lysosomal degradation pathways remain incompletely understood. Here, we identified a previously uncharacterized mammalian-specific protein, Beclin 2, which, like Beclin 1, functions in autophagy and interacts with class III PI3K complex components and Bcl-2. However, Beclin 2, but not Beclin 1, functions in an additional lysosomal degradation pathway. Beclin 2 is required for ligand-induced endolysosomal degradation of several G protein-coupled receptors (GPCRs) through its interaction with GASP1. Beclin 2 homozygous knockout mice have decreased embryonic viability, and heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, and obesity and insulin resistance. Our findings identify Beclin 2 as a converging regulator of autophagy and GPCR turnover and highlight the functional and mechanistic diversity of Beclin family members in autophagy, endolysosomal trafficking, and metabolism.

A Balancing Act: Learning from the Past to Build a Future-Focused Opioid Strategy.

The harmful side effects of opioid drugs such as respiratory depression, tolerance, dependence, and abuse potential have limited the therapeutic utility of opioids for their entire clinical history. However, no previous attempt to develop effective pain drugs that substantially ameliorate these effects has succeeded, and the current opioid epidemic affirms that they are a greater hindrance to the field of pain management than ever. Recent attempts at new opioid development have sought to reduce these side effects by minimizing engagement of the regulatory protein arrestin-3 at the mu-opioid receptor, but there is significant controversy around this approach. Here, we discuss the ongoing effort to develop safer opioids and its relevant historical context. We propose a new model that reconciles results previously assumed to be in direct conflict to explain how different signaling profiles at the mu-opioid receptor contribute to opioid tolerance and dependence. Our goal is for this framework to inform the search for a new generation of lower liability opioid analgesics.

Loss of D2 dopamine receptor function modulates cocaine-induced glutamatergic synaptic potentiation in the ventral tegmental area.

Potentiation of glutamate responses is a critical synaptic response to cocaine exposure in ventral tegmental area (VTA) neurons. However, the mechanism by which cocaine exposure promotes potentiation of NMDA receptors (NMDARs) and subsequently AMPA receptors (AMPARs) is not fully understood. In this study we demonstrate that repeated cocaine treatment causes loss of D2 dopamine receptor functional responses via interaction with lysosome-targeting G-protein-associated sorting protein1 (GASP1). We also show that the absence of D2 downregulation in GASP1-KO mice prevents cocaine-induced potentiation of NMDAR currents, elevation of the AMPA/NMDA ratio, and redistribution of NMDAR and AMPAR subunits to the membrane. As a pharmacological parallel, coadministration of the high-affinity D2 agonist, aripiprazole, reduces not only functional downregulation of D2s in response to cocaine but also potentiation of NMDAR and AMPAR responses in wild-type mice. Together these data suggest that functional loss of D2 receptors is a critical mechanism mediating cocaine-induced glutamate plasticity in VTA neurons.

Gut dysbiosis was inevitable, but tolerance was not: temporal responses of the murine microbiota that maintain its capacity for butyrate production correlate with sustained antinociception to chronic voluntary morphine.

The therapeutic benefits of opioids are compromised by the development of analgesic tolerance, which necessitates higher dosing for pain management thereby increasing the liability for dependence and addiction. Rodent models indicate opposing roles of the gut microbiota in tolerance: morphine-induced gut dysbiosis exacerbates tolerance, whereas probiotics ameliorate tolerance. Not all individuals develop tolerance which could be influenced by differences in microbiota, and yet no study has capitalized upon this natural variation to identify specific features linked to tolerance. We leveraged this natural variation in a murine model of voluntary oral morphine self-administration to elucidate the mechanisms by which microbiota influences tolerance. Although all mice shared similar and predictive morphine-driven microbiota changes that largely masked informative associations with variability in tolerance, our high-resolution temporal analyses revealed a divergence in the progression of dysbiosis that best explained differences in the development in tolerance. Mice that did not develop tolerance also maintained a higher abundance of taxa capable of producing the short-chain fatty acid (SCFA) butyrate, known to bolster intestinal barriers, suppress inflammation, and promote neuronal homeostasis. Furthermore, dietary butyrate supplementation significantly reduced the development of tolerance. These findings could inform immediate therapies to extend the analgesic efficacy of opioids.

Deletion of arrestin-3 does not improve compulsive drug-seeking behavior in a longitudinal paradigm of oral morphine self-administration.

Opioid drugs are potent analgesics that mimic the endogenous opioid peptides, endorphins and enkephalins, by activating the µ-opioid receptor. Opioid use is limited by side effects, including significant risk of opioid use disorder. Improvement of the effect/side effect profile of opioid medications is a key pursuit of opioid research, yet there is no consensus on how to achieve this goal. One hypothesis is that the degree of arrestin-3 recruitment to the µ-opioid receptor impacts therapeutic utility. However, it is not clear whether increased or decreased interaction of the µ-opioid receptor with arrestin-3 would reduce compulsive drug-seeking. To examine this question, we utilized three genotypes of mice with varying abilities to recruit arrestin-3 to the µ-opioid receptor in response to morphine in a novel longitudinal operant self-administration model. We demonstrate that arrestin-3 knockout and wild type mice have highly variable drug-seeking behavior with few genotype differences. In contrast, in mice where the µ-opioid receptor strongly recruits arrestin-3, drug-seeking behavior is much less varied. We created a quantitative method to define compulsivity in drug-seeking and found that mice lacking arrestin-3 were more likely to meet the criteria for compulsivity whereas mice with enhanced arrestin-3 recruitment did not develop a compulsive phenotype. Our data suggest that opioids that engage both G protein and arrestin-3, recapitulating the endogenous signaling pattern, will reduce abuse liability.

Trafficking properties of the D5 dopamine receptor.

Dopamine receptors are important for diverse biological functions and are important pharmacological targets in human medicine. Signal transduction from the dopamine receptors is controlled at many levels, including by the process of receptor trafficking. Little is known regarding the endocytic and postendocytic trafficking properties of the D5 dopamine receptor. Here, we show that endocytosis of the D5 receptor can be achieved both homologously, through direct receptor activation by agonist, and also heterologously, due to independent activation of protein kinase C (PKC). In contrast, the D1 receptor is endocytosed only in response to agonist but not PKC activation. We have identified the residue in the third intracellular loop of the D5 receptor that is both necessary for PKC-mediated endocytosis of the D5 receptor and sufficient to induce PKC-mediated endocytosis when introduced to the D1 receptor. In addition, we show that endocytosis of D5 through both pathways is dependent on clathrin and dynamin but that only agonist-induced endocytosis engages ß-arrestin 2. Together, these data show that the D5 receptor shows a trafficking profile distinct from that of any of the other dopamine receptors.

Novel screening assay for the selective detection of G-protein-coupled receptor heteromer signaling.

Drugs targeting G-protein-coupled receptors (GPCRs) make up more than 25% of all prescribed medicines. The ability of GPCRs to form heteromers with unique signaling properties suggests an entirely new and unexplored pool of drug targets. However, current in vitro assays are ill equipped to detect heteromer-selective compounds. We have successfully adapted an approach, using fusion proteins of GPCRs and chimeric G proteins, to create an in vitro screening assay (in human embryonic kidney cells) in which only activated heteromers are detectable. Here we show that this assay can demonstrate heteromer-selective G-protein bias as well as measure transinhibition. Using this assay, we reveal that the δ-opioid receptor agonist ADL5859, which is currently in clinical trials, has a 10-fold higher potency against δ-opioid receptor homomers than δ/µ-opioid receptor heteromers (pEC(50) = 6.7 ± 0.1 versus 5.8 ± 0.2). The assay enables the screening of large compound libraries to identify heteromer-selective compounds that could then be used in vivo to determine the functional role of heteromers and develop potential therapeutic agents.

Arrestin-3 Agonism at Dopamine D3 Receptors Defines a Subclass of Second-Generation Antipsychotics That Promotes Drug Tolerance.

BACKGROUND: Second-generation antipsychotics (SGAs) are frontline treatments for serious mental illness. Often, individual patients benefit only from some SGAs and not others. The mechanisms underlying this unpredictability in treatment efficacy remain unclear. All SGAs bind the dopamine D3 receptor (D3R) and are traditionally considered antagonists for dopamine receptor signaling. METHODS: Here, we used a combination of two-photon calcium imaging, in vitro signaling assays, and mouse behavior to assess signaling by SGAs at D3R. RESULTS: We report that some clinically important SGAs function as arrestin-3 agonists at D3R, resulting in modulation of calcium channels localized to the site of action potential initiation in prefrontal cortex pyramidal neurons. We further show that chronic treatment with an arrestin-3 agonist SGA, but not an antagonist SGA, abolishes D3R function through postendocytic receptor degradation by GASP1 (G protein-coupled receptor-associated sorting protein-1). CONCLUSIONS: These results implicate D3R-arrestin-3 signaling as a source of SGA variability, highlighting the importance of including arrestin-3 signaling in characterizations of drug action. Furthermore, they suggest that postendocytic receptor trafficking that occurs during chronic SGA treatment may contribute to treatment efficacy.

The G-protein coupled receptor associated sorting protein GASP-1 regulates the signalling and trafficking of the viral chemokine receptor US28.

Human cytomegalovirus (HCMV) encodes the seven transmembrane(7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Galphaq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-kappaB (NF-kappaB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.

Dopamine D(3) receptors are down-regulated following heterologous endocytosis by a specific interaction with G protein-coupled receptor-associated sorting protein-1.

The D(3) dopamine receptor is endocytosed through a heterologous mechanism mediated by phorbol esters. Here, we show that following this endocytosis the D(3) dopamine receptors fail to recycle and are instead targeted for degradation through an interaction with the G protein-coupled receptor (GPCR)-associated sorting protein-1 (GASP-1). Furthermore, we identified a specific binding motif in the C terminus common to the D(3) and D(2) that confers GASP-1 binding. shRNA knockdown of GASP-1 delayed post-endocytic degradation of both the D(2) and D(3) dopamine receptors. In addition, mutation of the D(2) and D(3) receptor C termini to resemble the D(4), which does not interact with GASP-1, not only inhibited GASP-1 binding but slowed degradation after endocytosis. Conversely, mutation of the C terminus of the D(4) to resemble that of the D(2) and D(3) facilitated GASP-1 binding and promoted post-endocytic degradation of the mutant D(4) receptor. Thus, we have identified a motif that is both necessary and sufficient to promote GASP-1 binding and receptor degradation. In addition, these data demonstrated that GASP-1 can mediate post-endocytic degradation of dopamine receptors that have been endocytosed not only as a consequence of dopamine activation but also as a consequence of activation by phorbol esters.

The chemoselective reactions of tyrosine-containing G-protein-coupled receptor peptides with [Cp*Rh(H2O)3](OTf)2, including 2D NMR structures and the biological consequences.

The bioconjugation of organometallic complexes with peptides has proven to be a novel approach for drug discovery. We report the facile and chemoselective reaction of tyrosine-containing G-protein-coupled receptor (GPCR) peptides with [Cp*Rh(H(2)O)(3)](OTf)(2), in water, at room temperature, and at pH 5-6. We have focused on three important GPCR peptides; namely, [Tyr(1)]-leu-enkephalin, [Tyr(4)]-neurotensin(8-13), and [Tyr(3)]-octreotide, each of which has a different position for the tyrosine residue, together with competing functionalities. Importantly, all other functional groups present, i.e., amino, carboxyl, disulfide, phenyl, and indole, were not prominent sites of reactivity by the Cp*Rh tris aqua complex. Furthermore, the influence of the Cp*Rh moiety on the structure of [Tyr(3)]-octreotide was characterized by 2D NMR, resulting in the first representative structure of an organometallic-peptide complex. The biological consequences of these Cp*Rh-peptide complexes, with respect to GPCR binding and growth inhibition of MCF7 and HT29 cancer cells, will be presented for [(η(6)-Cp*Rh-Tyr(1))-leu-enkephalin](OTf)(2) and [(η(6)-Cp*Rh-Tyr(3))-octreotide](OTf)(2).

Chronic methadone treatment shows a better cost/benefit ratio than chronic morphine in mice.

Chronic treatment of pain with opiate drugs can lead to analgesic tolerance and drug dependence. Although all opiate drugs can promote tolerance and dependence in practice, the severity of those unwanted side effects differs depending on the drug used. Although each opiate drug has its own unique set of pharmacological profiles, methadone is the only clinically used opioid drug that produces substantial receptor endocytosis at analgesic doses. Here, we examined whether moderate doses of methadone carry any benefits over chronic use of equianalgesic morphine, the prototypical opioid. Our data show that chronic administration of methadone produces significantly less analgesic tolerance than morphine. Furthermore, we found significantly reduced precipitated withdrawal symptoms after chronic methadone treatment than after chronic morphine treatment. Finally, using a novel animal model with a degrading µ-opioid receptor we showed that, although endocytosis seems to protect against tolerance development, endocytosis followed by receptor degradation produces a rapid onset of analgesic tolerance to methadone. Together, these data indicated that opioid drugs that promote receptor endocytosis and recycling, such as methadone, may be a better choice for chronic pain treatment than morphine and its derivatives that do not.

Functional characterization of human variants of the mu-opioid receptor gene.

Opioids and their receptors have an important role in analgesia and alcohol and substance use disorders (ASUD). We have identified several naturally occurring amino acid changing variants of the human mu-opioid receptor (MOR), and assessed the functional consequences of these previously undescribed variants in stably expressing cell lines. Several of these variants had altered trafficking and signaling properties. We found that an L85I variant showed significant internalization in response to morphine, in contrast to the WT MOR, which did not internalize in response to morphine. Also, when L85I and WT receptor were coexpressed, WT MOR internalized with the L85I MOR, suggesting that, in the heterozygous condition, the L85I phenotype would be dominant. This finding is potentially important, because receptor internalization has been associated with development of tolerance to opiate analgesics. In contrast, an R181C variant abolished both signaling and internalization in response to saturating doses of the hydrolysis-resistant enkephalin [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO). Coexpression of the R181C and WT receptor led to independent trafficking of the 2 receptors. S42T and C192F variants showed a rightward shift in potency of both morphine and DAMGO, whereas the S147C variant displayed a subtle leftward shift in morphine potency. These data suggest that these and other such variants may have clinical relevance to opioid responsiveness to both endogenous ligands and exogenous drugs, and could influence a broad range of phenotypes, including ASUD, pain responses, and the development of tolerance to morphine.

Opioid-Induced GABA potentiation after chronic morphine attenuates the rewarding effects of opioids in the ventral tegmental area.

GABA transmission in the ventral tegmental area (VTA) is critical for fine tuning the activity of dopamine neurons in response to opioids. However, the precise mechanism by which GABA input shapes opioid reward is poorly understood. We observed a reduction of conditioned place preference for low doses of the opioid [d-Ala2, N-MePhe4, Gly5-ol]-enkephalin (DAMGO) and a switch in the functional effects of µ-opioid receptor modulation of GABA postsynaptic currents in the mouse VTA 1 d after chronic morphine treatment. Specifically, whereas in naive mice DAMGO inhibits GABA postsynaptic currents, GABAergic currents are potentiated by DAMGO after chronic morphine treatment. Importantly, pretreatment with the cAMP signaling inhibitor (R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium both restored DAMGO reward and reversed the DAMGO-mediated potentiation, thereby reestablishing the inhibitory effects of opioids on GABA currents. Thus, a paradoxical bidirectionality in µ-receptor-mediated control of GABA transmission following chronic morphine treatment is a critical mechanism that determines the expression of opioid reward in the VTA.

micro-Opioid receptor endocytosis prevents adaptations in ventral tegmental area GABA transmission induced during naloxone-precipitated morphine withdrawal.

Chronic morphine drives adaptations in synaptic transmission thought to underlie opiate dependence. Here we examine the role of micro-opioid receptor (MOR) trafficking in one of these adaptations, specifically, changes in GABA transmission in the ventral tegmental area (VTA). To address this question, we used a knock-in mouse, RMOR (for recycling MOR), in which genetic change in the MOR promotes morphine-induced receptor desensitization and endocytosis in GABA interneurons of the VTA. In wild-type mice (postnatal days 23-28) chronic morphine (10 mg/kg, s.c., twice daily for 5 d), induced a cAMP-dependent increase in the probability of GABA release onto VTA dopamine neurons. The increased GABA release frequency correlated with physical dependence on morphine measured by counting somatic signs of morphine withdrawal, such as, tremors, jumps, rears, wet-dog shakes, and grooming behavior precipitated by subcutaneous administration of naloxone (NLX) (2 mg/kg). This adaptation in GABA release was prevented in RMOR mice given the same morphine treatment, implicating MOR trafficking in this morphine-induced change in plasticity. Importantly, treatment with the cAMP activity inhibitor rp-cAMPS [(R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium] (50 ng/0.5 microl), directly to the VTA, attenuated somatic withdrawal signs to systemic morphine produced by intra-VTA NLX (500 ng/0.5 microl), directly tying enhanced cAMP-driven GABA release to naloxone-precipitated morphine withdrawal in the VTA.

Morphine-induced receptor endocytosis in a novel knockin mouse reduces tolerance and dependence.

Opioid drugs, such as morphine, are among the most effective analgesics available. However, their utility for the treatment of chronic pain is limited by side effects including tolerance and dependence. Morphine acts primarily through the mu-opioid receptor (MOP-R) , which is also a target of endogenous opioids. However, unlike endogenous ligands, morphine fails to promote substantial receptor endocytosis both in vitro, and in vivo. Receptor endocytosis serves at least two important functions in signal transduction. First, desensitization and endocytosis act as an "off" switch by uncoupling receptors from G protein. Second, endocytosis functions as an "on" switch, resensitizing receptors by recycling them to the plasma membrane. Thus, both the off and on function of the MOP-R are altered in response to morphine compared to endogenous ligands. To examine whether the low degree of endocytosis induced by morphine contributes to tolerance and dependence, we generated a knockin mouse that expresses a mutant MOP-R that undergoes morphine-induced endocytosis. Morphine remains an excellent antinociceptive agent in these mice. Importantly, these mice display substantially reduced antinociceptive tolerance and physical dependence. These data suggest that opioid drugs with a pharmacological profile similar to morphine but the ability to promote endocytosis could provide analgesia while having a reduced liability for promoting tolerance and dependence.

Heteromerization of the µ- and δ-opioid receptors produces ligand-biased antagonism and alters µ-receptor trafficking.

Heteromerization of opioid receptors has been shown to alter opioid receptor pharmacology. However, how receptor heteromerization affects the processes of endocytosis and postendocytic sorting has not been closely examined. This question is of particular relevance for heteromers of the µ-opioid receptor (MOR) and δ-opioid receptor (DOR), because the MOR is recycled primarily after endocytosis and the DOR is degraded in the lysosome. Here, we examined the endocytic and postendocytic fate of MORs, DORs, and DOR/MOR heteromers in human embryonic kidney 293 cells stably expressing each receptor alone or coexpressing both receptors. We found that the clinically relevant MOR agonist methadone promotes endocytosis of MOR but also the DOR/MOR heteromer. Furthermore, we show that DOR/MOR heteromers that are endocytosed in response to methadone are targeted for degradation, whereas MORs in the same cell are significantly more stable. It is noteworthy that we found that the DOR-selective antagonist naltriben mesylate could block both methadone- and [D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin-induced endocytosis of the DOR/MOR heteromers but did not block signaling from this heteromer. Together, our results suggest that the MOR adopts novel trafficking properties in the context of the DOR/MOR heteromer. In addition, they suggest that the heteromer shows "biased antagonism," whereby DOR antagonist can inhibit trafficking but not signaling of the DOR/MOR heteromer.

A novel knock-in mouse reveals mechanistically distinct forms of morphine tolerance.

The role of µ-opioid receptor (MOR) down-regulation in opioid tolerance remains controversial. In this study, we used a novel knock-in mouse to examine how changing the extent of MOR down-regulation alters the development of morphine tolerance. These mice express a mutant MOR, degrading MOR (DMOR), that differs from the wild-type (WT) MOR in two ways: 1) unlike the recycling WT MOR, the mutant DMOR is targeted for degradation after its internalization, thus facilitating down-regulation; and 2) unlike the WT MOR, DMOR is efficiently internalized in response to morphine activation. We found that both WT MOR and DMOR mice develop tolerance to morphine, but DMOR mice exhibit a more rapid onset of tolerance and show receptor down-regulation. WT MOR mice develop morphine tolerance more slowly but even once profoundly tolerant show no receptor down-regulation. Furthermore, WT mice show significantly more morphine dependence than DMOR mice after long-term treatment as indicated by withdrawal. Taken together these data indicate that tolerance mediated by receptor down-regulation manifests differently both at the behavioral and biochemical level than does the actual morphine tolerance that occurs in WT mice and that loss of receptor function is not a major contributor to morphine tolerance in WT MOR mice.

How to design an opioid drug that causes reduced tolerance and dependence.

Mu opioid receptor (MOR) agonists such as morphine are extremely effective treatments for acute pain. In the setting of chronic pain, however, their long-term utility is limited by the development of tolerance and physical dependence. Drug companies have tried to overcome these problems by simply "dialing up" signal transduction at the receptor, designing more potent and efficacious agonists and more long-lasting formulations. Neither of these strategies has proven to be successful, however, because the net amount of signal transduction, particularly over extended periods of drug use, is a product of much more than the pharmacokinetic properties of potency, efficacy, half-life, and bioavailability, the mainstays of traditional pharmaceutical screening. Both the quantity and quality of signal transduction are influenced by many regulated processes, including receptor desensitization, trafficking, and oligomerization. Importantly, the efficiency with which an agonist first stimulates signal transduction is not necessarily related to the efficiency with which it stimulates these other processes. Here we describe recent findings that suggest MOR agonists with diminished propensity to cause tolerance and dependence can be identified by screening drugs for the ability to induce MOR desensitization, endocytosis, and recycling. We also discuss preliminary evidence that heteromers of the delta opioid receptor and the MOR are pronociceptive, and that drugs that spare such heteromers may also induce reduced tolerance.

Pharmacological and genetic manipulations at the µ-opioid receptor reveal arrestin-3 engagement limits analgesic tolerance and does not exacerbate respiratory depression in mice.

Opioid drugs are widely used analgesics that activate the G protein-coupled µ-opioid receptor, whose endogenous neuropeptide agonists, endorphins and enkephalins, are potent pain relievers. The therapeutic utility of opioid drugs is hindered by development of tolerance to the analgesic effects, requiring dose escalation for persistent pain control and leading to overdose and fatal respiratory distress. The prevailing hypothesis is that the intended analgesic effects of opioid drugs are mediated by µ-opioid receptor signaling to G protein, while the side-effects of respiratory depression and analgesic tolerance are caused by engagement of the receptor with the arrestin-3 protein. Consequently, opioid drug development has focused exclusively on identifying agonists devoid of arrestin-3 engagement. Here, we challenge the prevailing hypothesis with a panel of six clinically relevant opioid drugs and mice of three distinct genotypes with varying abilities to promote morphine-mediated arrestin-3 engagement. With this genetic and pharmacological approach, we demonstrate that arrestin-3 recruitment does not impact respiratory depression, and effective arrestin-3 engagement reduces, rather than exacerbates, the development of analgesic tolerance. These studies suggest that future development of safer opioids should focus on identifying opioid ligands that recruit both G protein and arrestin-3, thereby mimicking the signaling profile of most endogenous µ-opioid receptor agonists.